Nano-liposome as well as preparation method and application thereof

Abstract

The invention discloses a nano-liposome and a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations, the preparation method comprises the following steps: S1, mixing soybean lecithin, cholesterol and Tween 80, dissolving in absolute ethyl alcohol, stirring until complete dissolution, and transferring the mixed solution into a rotary evaporator to obtain a lipid film; and S2, adding a resveratrol solution, a nisin solution and a 0.03 M phosphate buffer solution into the lipid membrane obtained in the step S1, after the membrane is completely dissolved, transferring to a cell superfine grinding instrument for treatment, adding a phosphatidyl ethanolamine-polyethylene glycol 2000 aqueous solution with the mass fraction of 3%, uniformly mixing by vortex, standing, and filtering by a filtering membrane to obtain the lipid membrane. The polyethylene glycol modified nano liposome RN-NPs is obtained. As an oral targeted drug delivery system, the nano-liposome disclosed by the invention not only improves the effective utilization rate of drugs, but also remarkably improves the safety and effectiveness of treating staphylococcus aureus enteritis.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and specifically relates to a nano liposome and a preparation method and application thereof. Background Art

[0002] In the post-antibiotic era, antibiotic resistance has become a global public health crisis. The prevalence of multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) poses a major challenge to traditional antibiotic treatment. MRSA can cause serious infections, including antibiotic-associated diarrhea (AAD) and antibiotic-associated enterocolitis (AAE), for which treatment options are very limited and the treatment effects are often unsatisfactory.

[0003] To meet this challenge, researchers have begun to explore new antimicrobial therapies, especially the development of antimicrobial drug delivery systems that can target specific pathogens. These systems are designed to improve the bioavailability of drugs, reduce the development of drug resistance, and reduce interference with normal flora. The application of nanotechnology has brought revolutionary progress in this field, especially nanoliposomes, as a biocompatible and targeted nanocarrier, have become a hot topic of research.

[0004] Nanoliposomes are small vesicles composed of phospholipid bilayers that can encapsulate hydrophilic and lipophilic drugs, protect them from damage by the external environment, and provide a way to control drug release. As a new type of drug delivery system, nanoliposomes have shown great potential in improving drug efficacy and reducing side effects, but they face challenges in stability, size control, drug release, biocompatibility, large-scale production, targeting, clinical transformation, and regulatory supervision. Summary of the invention

[0005] The purpose of the present invention is to provide a nanoliposome and a preparation method and application thereof. The nanoliposome, as an oral targeted drug delivery system, not only improves the effective utilization rate of drugs, but also significantly improves the safety and effectiveness of treating Staphylococcus aureus enteritis.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing nanoliposomes comprises the following steps:

[0008] S1. Dissolve soybean lecithin, cholesterol and Tween 80 in anhydrous ethanol, stir until completely dissolved, and transfer the mixed solution to a rotary evaporator to obtain a lipid film;

[0009] S2, adding resveratrol (Res) solution, nisin (Nisin) solution and 0.03M phosphate buffer to the lipid film obtained in step S1, and transferring the film to a cell ultrafine pulverizer for treatment to obtain a nanoliposome solution after the film is completely dissolved;

[0010] S3. Add 3% by mass of phosphatidylethanolamine-polyethylene glycol 2000 aqueous solution to the nanoliposome solution obtained in step S2, vortex and mix evenly, let stand, and filter with a filter membrane to obtain polyethylene glycol-modified nanoliposome RN-NPs.

[0011] Preferably, in step S1, the weight ratio of soybean lecithin, cholesterol and Tween 80 is 6:1:1.8.

[0012] Preferably, in step S1, 1.2 g soybean lecithin, 0.2 g cholesterol, and 0.36 g Tween 80 are mixed and dissolved in 50 mL of anhydrous ethanol.

[0013] Preferably, in step S1, the mixed solution is transferred to a rotary evaporator and heated in a water bath at 50-60° C. for 5-15 min to obtain a lipid film.

[0014] Preferably, in step S2, the pH of the phosphate buffer is 7.2-7.4.

[0015] Preferably, in step S2, the cell ultrafine pulverizer processing parameters are: working for 10 seconds, interval of 5 seconds, working for 30 minutes at a power of 300W.

[0016] Preferably, in step S2, after vortexing to mix evenly, the mixture is allowed to stand at 4° C. for 1 h.

[0017] Preferably, in step S2, the filter membrane is a 0.22 mm microporous filter membrane.

[0018] The invention also provides a method for preparing the nano liposomes obtained by the preparation method.

[0019] The present invention also provides a nanoliposome prepared by the preparation method or application of the nanoliposome in preparing a product for targeted treatment of Staphylococcus aureus enteritis.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The present invention discloses a nanoliposome, which effectively protects Res and Nisin from degradation by gastric acid and protease, and avoids premature release and absorption of drugs in the digestive tract. At the same time, the unique phospholipid bilayer structure of RN-NPs can react specifically with the pore-forming toxins released by Staphylococcus aureus to form a pore structure, so that the drug can be released in a targeted manner at the site of intestinal inflammation. This not only improves the effective utilization rate of Res and Nisin, but also significantly improves the safety and effectiveness of treatment, and reduces the side effects caused by non-targeted treatment.

[0022] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The size and potential results of RN-NPs and blank liposomes provided in Example 1 and Comparative Example 1, respectively, wherein: Figure 1 A in is the average particle size and frequency distribution result, Figure 1 B in the figure is the PDI and Zeta results;

[0024] Figure 2 The HPLC chromatogram of RN-NPs provided in Example 1, wherein: Figure 2 A in the figure is the HPLC chromatogram of Res and Nisin standard products. Figure 2 B in the figure is the HPLC chromatogram of RN-NPs and RN-NPs after incubation with ethanol;

[0025] Figure 3 The FTIR image and CLSM image of RN-NPs provided in Example 1, wherein: Figure 3 A in the figure is the FTIR image. Figure 3 B in the figure is the CLSM image;

[0026] Figure 4 The stability and digestion simulation results of RN-NPs provided in Example 1, wherein Figure 4 A in the figure is the change of average particle size, PDI and Zeta potential of RN-NPs at 4℃, 22℃ and 37℃. Figure 4 B in the figure is the hydrolytic stability result in simulated intestinal fluid;

[0027] Figure 5 The average particle size, PDI and Zeta potential change results of RN-NPs provided in Example 1 during SGF and SIF digestion, wherein: Figure 5 A in the figure is the average particle size change result. Figure 5 B in the figure is the result of PDI change. Figure 5 C in is the result of Zeta potential change;

[0028] Figure 6 The microstructure changes of RN-NPs provided in Example 1 during SGF and SIF digestion, wherein: Figure 6 A is 0h in the SGF microstructure, Figure 6 B is 2h in the SGF microstructure, Figure 6 C is 4h in the SGF microstructure, Figure 6 The D is 0h in the SIF microstructure, Figure 6 E is 2h in SIF microstructure, Figure 6 The F in the figure is 4h in SIF microstructure;

[0029] Figure 7 The plate colonies after incubation of RN-NPs provided in Example 1 with Staphylococcus aureus or Escherichia coli, wherein: Figure 7 A in the figure is the colony of Staphylococcus aureus on the plate at 0h. Figure 7 B in the figure is the 24h plate colony of Staphylococcus aureus. Figure 7 C in the figure is the colony of Staphylococcus aureus on the plate after 48 hours. Figure 7 D in the figure is the colony of Staphylococcus aureus on the plate after 72 hours. Figure 7 E in the figure is the colony of RN-NPs and Staphylococcus aureus at 0h. Figure 7 F in the figure is the 24h plate colony situation of RN-NPs and Staphylococcus aureus. Figure 7 G in the figure is the colony of RN-NPs and Staphylococcus aureus on the plate after 48 hours. Figure 7 H in the figure is the colony of RN-NPs and Staphylococcus aureus on the plate at 72h. Figure 7 I in the figure is the colony of E. coli on the plate at 0h. Figure 7 J in the figure is the 24h plate colony of E. coli. Figure 7 K in the figure is the colony of E. coli on the plate after 48 hours. Figure 7 L in the figure is the colony of E. coli on the plate after 72 hours. Figure 7 M in the figure is the colony of RN-NPs and E. coli at 0h. Figure 7 N is the 24h plate colony situation of RN-NPs and E. coli. Figure 7 The O in the figure is the colony of RN-NPs and E. coli on the plate after 48 hours. Figure 7 P in the figure is the colony situation of RN-NPs and Escherichia coli on the plate at 72h;

[0030] Figure 8 The HPLC chromatograms of RN-NPs, RN-NPs incubated with Escherichia coli, and RN-NPs incubated with Staphylococcus aureus provided in Example 1;

[0031] Fig. 9 The results of the cell viability study of the RN-NPs provided in Example 1 and the blank liposomes provided in Comparative Example 1, wherein: Fig. 9 A in the figure is the protein content of the RN-NPs provided in Example 1 and the blank liposomes provided in Comparative Example 1 after co-incubation with LPS-stimulated J774A.1 cells. Fig. 9 B in the figure is the change of TNF-α content after co-incubation of RN-NPs provided in Example 1 and blank liposomes provided in Comparative Example 1 with LPS-stimulated J774A.1 cells;

[0032] Fig.10 The cell survival rate of RN-NPs provided in Example 1 after incubation with J774A.1 mouse macrophages and the hemolytic test results with mouse blood, wherein, Fig.10 A in the figure is the cell survival rate after incubation of RN-NPs with J774A.1 mouse macrophages. Fig.10 B in the figure is the result of the hemolytic test of RN-NPs and mouse blood;

[0033] Fig.11 The modeling and experimental flow chart of the RN-NPs of the present invention for treating Staphylococcus aureus enteritis in mice;

[0034] Fig.12 The results of the in vivo efficacy study on mice after taking the antibiotic mixed solution, the positive drug oxacillin sodium, free RN, and RN-NPs of Example 1, wherein: Fig.12 A in the figure is the weight result of mice. Fig.12 B in the table is the disease activity index (DAI) score result. Fig.12 C in the figure is the statistical chart of inflammatory factor INF-γ. Fig.12 D in the figure is the IL-6 statistical chart. Fig.12 E in the figure is the statistical diagram of MPO activity in colon tissue;

[0035] Fig.13 The results of the treatment of enteritis by RN-NPs, blank group, model group, positive control group, antibiotic group, and free RN group provided in Example 1, wherein: Fig.13 A in the figure is the hematoxylin and eosin (H&E) stained section of the colon tissue of mice in the blank group. Fig.13 B is the hematoxylin and eosin (H&E) stained section of the colon tissue of mice in the model group. Fig.13 C in the figure is a hematoxylin and eosin (H&E) stained section of the colon tissue of mice in the positive control group. Fig.13 D in the figure is a partial enlarged view of the H&E-stained section of the colon tissue of mice in the blank group. Fig.13 E in the figure is a partial enlarged view of the hematoxylin and eosin (H&E) stained section of the colon tissue of mice in the model group. Fig.13 F in the figure is a partial magnified image of the hematoxylin and eosin (H&E) stained section of the colon tissue of mice in the positive control group. Fig.13 G in the figure is the hematoxylin and eosin (H&E) stained section of the colon tissue of mice in the antibiotic group. Fig.13 H in the figure is the hematoxylin and eosin (H&E) stained section of the colon tissue of mice in the free RN group. Fig.13 I in the figure is the hematoxylin and eosin (H&E) stained section of the colon tissue of mice in the RN-NPs group. Fig.13 J in the figure is a partial enlarged view of the hematoxylin and eosin (H&E) stained section of the colon tissue of mice in the antibiotic group. Fig.13 K in the figure is a partial enlarged view of the hematoxylin and eosin (H&E) stained section of the colon tissue of mice in the free RN group. Fig.13 L in the figure is a partial magnified image of the hematoxylin and eosin (H&E) stained section of the colon tissue of mice in the RN-NPs group;

[0036] Fig.14 The histological scores of RN-NPs, blank group, model group, positive control group, antibiotic group, and free RN group provided in Example 1;

[0037] Fig.15 The in vivo safety evaluation results of RN-NPs in the Staphylococcus aureus enteritis test are shown in Figure 2. Fig.15 A in the figure is the heart section of mice in the control group. Fig.15 B in the figure is the heart section of the model group mice. Fig.15 C in the figure is the heart section of mice in the positive control group. Fig.15 D in the figure is the heart section of mice in the antibiotic group. Fig.15 E in the figure is the heart slice of mice in the free RN group. Fig.15 F in the figure is the heart slice of mice in the RN-NPs group. Fig.15 G in the figure is the liver section of mice in the control group. Fig.15 H in the figure is the liver section of mice in the model group. Fig.15 I in the figure is the liver section of mice in the positive control group, Fig.15 J in the figure is the liver section of mice in the antibiotic group. Fig.15 K in the figure is the liver section of mice in the free RN group. Fig.15 L in the figure is the liver section of mice in the RN-NPs group. Fig.15 M in the figure is the spleen section of mice in the control group. Fig.15 N in the figure is the spleen section of mice in the model group. Fig.15 O in the figure is the spleen section of mice in the positive control group. Fig.15 P in the figure is the spleen section of mice in the antibiotic group. Fig.15 Q in the figure is the spleen section of mice in the free RN group. Fig.15R in the figure is the spleen section of mice in the RN-NPs group. Fig.15 The S in the figure is the kidney section of the mice in the control group. Fig.15 T in the figure is the kidney section of the model group mice. Fig.15 U in the figure is the kidney section of mice in the positive control group. Fig.15 V in the figure is the kidney section of mice in the antibiotic group. Fig.15 W in the figure is the kidney section of mice in the free RN group. Fig.15 The X in the figure indicates the kidney sections of mice in the RN-NPs group. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0039] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0040] The experimental materials and reagents involved in the following examples are:

[0041] Methicillin-resistant Staphylococcus aureus ATCC 33591 and Escherichia coli O157 were both obtained from the laboratory of Qingdao Agricultural University.

[0042] Resveratrol (purity>99%), nisin type A (≥1000UI / mg), soybean lecithin (purity>98%), cholesterol (purity>98%), Tween 80 and other reagents were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0043] LB agar medium, brain heart infusion broth (BHI) medium, propidium iodide (PI), dimethyl sulfoxide (DMSO), pepsin and trypsin were purchased from Beijing Solebow Technology Co., Ltd.

[0044] DMEM high-glucose medium, bovine serum albumin (FBS), phosphate-buffered saline (PBS), 1% penicillin, 100 μg / mL streptomycin, and Hank’s balanced salt solution were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0045] J774A.1 mouse macrophages were obtained from the National Certified Cell Culture Collection (Shanghai, China). SPF male Kunming mice (5–6 weeks) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (License No.: SCXK 20220006). Mice were fed a standard rodent diet with free access to water and kept in cages at 22–25°C.

[0046] Other test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0047] Embodiment 1 This embodiment provides a nanoliposome, and the preparation method comprises the following steps:

[0048] S1. Dissolve 1.2 g soybean lecithin, 0.2 g cholesterol and 0.36 g Tween 80 in 50 mL anhydrous ethanol, stir until completely dissolved, transfer the mixed solution to a rotary evaporator, and heat in a water bath at 60°C for 5 min to obtain a lipid film;

[0049] S2, adding 20 mL of resveratrol solution, 20 mL of nisin type A solution and 0.03 M phosphate buffer with a pH of 7.2 to the lipid film obtained in step S1, and transferring the film to a cell ultrafine grinder for processing, working for 10 seconds each, with an interval of 5 seconds, and working for 30 minutes at a power of 300 W to obtain nanoliposomes;

[0050] S3. Add an equal volume of 3% by mass phosphatidylethanolamine-polyethylene glycol 2000 aqueous solution to the nanoliposome solution obtained in step S2, vortex and mix evenly, let stand at 4° C. for 1 h, and filter through a 0.22 mm microporous membrane to obtain polyethylene glycol-modified nanoliposomes (RN-NPs).

[0051] Comparative Example 1 This example provides a blank liposome, 1.2g soybean lecithin, 0.2g cholesterol, 0.36g Tween 80 are mixed and dissolved in 50mL of anhydrous ethanol, stirred until completely dissolved, and the mixed solution is transferred to a rotary evaporator, heated in a water bath at 60°C for 5min to obtain a lipid film. 50mL of 0.03M phosphate buffer with a pH of 7.2 is added thereto, and the film is completely dissolved, and then transferred to a cell superfine pulverizer for processing, working for 10s, with an interval of 5s, working for 30min at a power of 300W, and filtered with a 0.22mm microporous membrane to obtain a nano blank liposome.

[0052] The effects of Example 1 and Comparative Example 1 were verified by the following experiments:

[0053] 1. The average particle size, PDI and potential of liposomes were measured using a Malvern laser particle size analyzer. At 20°C, the refractive index of phospholipids (1.490) was 1.120 relative to the refractive index of the dispersion medium (1.330), and the absorbance of phospholipids was 0.001. All samples were diluted 10 times with ultrapure water, and each sample was measured in parallel more than three times. The results are as follows Figure 1 .

[0054] Depend on Figure 1From A in the figure, it can be seen that the average particle sizes of blank nanoliposomes and RN-NPs are 67.67±0.53nm and 146.50±0.83nm, respectively. It can be seen that the average particle size of RN-NPs provided in Example 1 is slightly larger than the average particle size of blank nanoliposomes provided in Comparative Example 1. The Zeta potential of blank nanoliposomes is -11.87±0.22mV, while the Zeta potential of RN-NPs is -14.61±1.03mV. The larger the absolute value of the Zeta potential, the stronger the charge on the surface of the particles, and the stronger the repulsion between the particles. It can be seen that RN-NPs have a stronger repulsive force, which can more effectively prevent liposome aggregation and make the system more stable. In addition, through Figure 1 Figure B shows that the PDI values ​​of blank nanoparticles and RN-NPs are both less than 0.3, which indicates that the liposomes are uniformly dispersed and stable.

[0055] 2. Prepare standard solutions of Res and Nisin and draw standard curves. Liposomes were separated by 30kda ultrafiltration tubes and filtered using 0.22μm filter membranes. An equal amount of ethanol demulsifier was added, ultrasonic treatment was performed at 25°C for 1 hour, the mixture was allowed to stand for 24 hours, and then filtered. The total concentration of Res and Nisin in the filtrate was determined by C18 column HPLC. The content of Res and Nisin in the sample and its encapsulation efficiency were calculated based on the peak area and standard curve. The results are shown in Figure 2. Figure 2 .

[0056] The embedding rates of Res and Nisin in RN-NPs were detected by high performance liquid chromatography (HPLC), and control experiments were performed to verify whether RN was successfully embedded. Figure 2 A and Figure 2 As shown in B. Figure 2 From A in the figure, we can see that the retention times of the characteristic peaks of Res and Nisin in the chromatogram are 12.20min and 16.03min, respectively. Figure 2 As shown in B, the characteristic peaks of Res and Nisin did not appear in the chromatogram, proving that there was no free Res and Nisin in the RN-NPs solution. In order to verify whether RN was successfully embedded in the liposomes, the RN-NPs solution was incubated with an equal amount of ethanol demulsifier to cause the liposome membrane to rupture and release the Res and Nisin embedded therein. Subsequently, the treated ethanol / RN-NPs sample was tested by HPLC again. Figure 2As shown in the red chromatogram of B in the figure, compared with the untreated RN-NPs control group, the characteristic peaks of Res and Nisin appeared at retention times of 12.21min and 15.89min, respectively, in the ethanol / RN-NPs group, and the peak areas of Res and Nisin increased significantly. This result shows that RN was successfully encapsulated in the liposomes and released after the liposome membrane was destroyed. Using the peak area changes of Res and Nisin before and after the destruction of RN-NPs, the standard curve and the encapsulation rate calculation formula, the encapsulation rate of Res was 35.45%, while the encapsulation rate of Nisin was 29.10%. This experiment successfully verified the encapsulation of Res and Nisin in RN-NPs, thus providing basic data support for its stability and effectiveness in practical applications.

[0057] 3. The RN-NPs provided in Example 1 were characterized by Fourier transform spectroscopy (FTIR) and confocal laser scanning microscopy (CLSM). The experimental scheme is as follows: The morphology of the liposomes was observed by CLSM. 40 μL of Nile red dye (0.25 mg / mL) was added to 500 μL of liposome sample and mixed evenly. 10 μL of sample was dropped onto the confocal microscope slide, semi-dried at room temperature, and covered with a coverslip. First use a low-power microscope to find the appropriate field of view and then observe with a high-power microscope. An FTIR spectrometer was used to determine the encapsulation of antibacterial substances by liposomes. The liposomes were freeze-dried. Then, Res, Nisin, blank liposomes, and RN-NPs were scanned to obtain FTIR spectra: using an MCT-A infrared detector with a resolution of 8 cm -1 , infrared spectrum scanning range is 4000-500cm -1 Each image was scanned 64 times. Figure 3 .

[0058] RN-NPs were characterized by FTIR and CLSM. Figure 3 A and Figure 3 From B, we can see that Res is between 2917-2995cm -1 There is an obvious hydroxyl absorption peak in the range. -1 and 1435.73cm -1 There are obvious ester group (C=O and CO) absorption peaks at 3628.96cm -1 Axial stretching of OH and NH was observed at 2933.69 cm -1 -NH 3+ The asymmetric stretching vibration of amide B is related to its specific vibration frequency. The lecithin in the wall material is at 1732.73 cm -1The stretching vibration of C=O in COO- is at 3382.42cm -1 There is a strong absorption peak at 2920.46cm, which is related to the NH stretching of Nisin and the stretching vibration of lecithin, indicating that the formation of liposomes forms hydrogen bonds between lecithin and Res or Nisin, thus shifting the absorption peak to the left. -1 The absorption peak at 2920.46cm proves that the stretching vibration of CH in Nisin moves to the low wave number direction due to the combination. -1 and 1733.24cm -1 The characteristic peak of Res was found at 40°. In summary, the analysis of Fourier transform infrared spectroscopy showed that Res, Nisin and phosphatidylcholine were mainly combined by hydrogen bonds and electrostatic interactions. The microstructure of liposomes was observed by CLSM. Figure 3 As shown in Figure B, the RN-NPs particles not only have a smooth surface and a regular spherical shape, but also have good dispersibility and no agglomeration. At the same time, the bioactive components embedded in the liposomes can also be clearly seen through the image.

[0059] 4. The RN-NPs nanoliposomes provided in Example 1 were subjected to stability and digestion simulation tests. The test scheme was as follows: the particle size, PDI, and Zeta potential of the RN-NPs stored at 4°C, 22°C, and 37°C for 7 days were measured to evaluate the stability of the liposomes.

[0060] In vitro digestion experiment: NaCl, HCl, K2HPO4, and NaOH were used to prepare simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The pH of SGF was adjusted to 1.2 with 0.1M HCl solution, and the pH of SIF was adjusted to 7.4 with 0.01M NaOH solution. The liposomes were mixed with SGF and SIF solutions at a ratio of 1:3 (v / v), and pepsin and trypsin were added to make the concentration 3.2mg / mL, and the mixture was kept in a constant temperature water bath at 37℃ and shaken evenly at 95rpm. Samples were taken for analysis at regular intervals of digestion time.

[0061] The in vitro lipolysis reaction of liposomes was determined by monitoring pH and automatic acid-base titration technology. Liposomes were mixed with SIF solution and the pH was adjusted to 7.4. 3.2 mg / mL trypsin and bile salts were added and reacted with stirring at a constant temperature of 37°C in a water bath. 0.01 M NaOH was used to adjust the pH value to 7.40 ± 0.01. A standard curve was drawn for the free fatty acid release of oleic acid under the same conditions.

[0062] In the digestion experiment, the liposomes were taken for a certain digestion time (0h, 2h, 4h) and the surface morphology was observed using an optical microscope. Figure 4-6 .

[0063] After characterizing the RN-NPs, the stability of the RN-NPs nanoliposomes provided in Example 1 was further analyzed. Figure 4 As shown in A, RN-NPs were stored at different temperatures (4°C, 22°C, and 37°C) for 7 days. The results showed that the average particle size and PDI of RN-NPs showed a trend of first decreasing and then increasing. The reason for this phenomenon may be that the hydrogen bonding ability of the liposome surface weakened at the beginning of storage, which reduced the average particle size. At the end of storage, the electrostatic interaction between liposomes weakened, and some liposomes aggregated, resulting in an increase in the average particle size and PDI. At the same time, compared with RN-NPs stored at 4°C, the average particle size of RN-NPs stored at 22°C and 37°C changed more significantly. This is because the thermal motion of molecules is affected by temperature. At the end of storage, the increased temperature accelerates the thermal motion of the particles, resulting in a decrease in electrostatic repulsion, thereby increasing collisions and aggregation between liposomes. Overall, RN-NPs have the smallest average particle size change and the best stability at 4°C.

[0064] In order to evaluate the stability of liposomes in simulated intestinal fluid (SIF), the hydrolysis behavior and stability of liposomes during digestion were detected by measuring the amount of free fatty acids released by crude liposomes and nanoliposomes in SIF. First, the free fatty acid content released by known concentrations of oleic acid (0-15000 μmol) under SIF conditions was used as a measure of hydrolysis. By measuring the amount of free fatty acids released by liposomes during digestion, the hydrolysis degree of crude liposomes and nanoliposomes was compared to evaluate their stability. The results are shown in Figure 2. Figure 4As shown in Figure B, the amount of free fatty acids released by both crude liposomes and nanoliposomes in SIF is much lower than the amount of free fatty acids released by oleic acid itself. This shows that the structure of liposomes can prevent or delay the release of free fatty acids to a certain extent. At the same time, compared with nanoliposomes, crude liposomes release more free fatty acids during the entire digestion process, which indicates that the hydrolysis rate of crude liposomes in SIF is higher. The main reason for this phenomenon may be that the surface area to volume ratio of crude liposomes is small, and there may be defects or irregular areas in their liposome membrane structure. These physical structural instabilities make crude liposomes more prone to deformation, aggregation or fusion, thereby increasing the release of free fatty acids. In contrast, nanoliposomes have a more compact and stable structure and are more resistant to hydrolysis reactions in SIF. Secondly, the amount of free fatty acids released by nanoliposomes has begun to stabilize in the early stage of digestion, while crude liposomes reach this stable state after a long time. In summary, the results showed that nanoliposomes showed higher stability in SIF, with significantly lower free fatty acid release than crude liposomes, and lower hydrolysis rates at the beginning of digestion. This stability can be attributed to the compactness and uniformity of the nanoliposome structure, which effectively resists the effects of hydrolysis during digestion. Therefore, nanoliposomes may provide better protection and release control in gastrointestinal applications, especially in drug delivery systems that require delayed release or improved stability.

[0065] The average particle size and PDI of RN-NPs vary as shown in Figure 5 A and Figure 5 As shown in B. The average particle size of RN-NPs changed little during the simulated gastric fluid (SGF) digestion stage, indicating that the liposome membrane of RN-NPs can effectively resist the effects of low pH and pepsin, allowing RN-NPs to pass through the stomach smoothly to the intestine. After entering the simulated intestinal fluid (SIF) digestion stage, the average particle size of RN-NPs began to decrease significantly and showed a continuous downward trend. This change is mainly attributed to the emulsification effect of bile salts in SIF on RN-NPs and the hydrolysis of the liposome membrane by trypsin. After the entire SIF digestion process, the average particle size of RN-NPs is still larger than that of blank nanoliposomes, indicating that RN-NPs still maintain a certain structural integrity in the intestinal environment. At the same time, as Figure 5 As shown in Figure B, the PDI of RN-NPs is relatively stable throughout the digestion stage, showing good dispersibility and uniformity. This indicates that RN-NPs can maintain a stable dispersion state in the simulated gastrointestinal environment and are not prone to significant aggregation or precipitation. Figure 5The C results in Figure 3 show that the Zeta potential of RN-NPs remains relatively stable in SGF, which may be due to the fact that the strong acidic environment of SGF and other factors have little effect on the surface charge of the liposome membrane. However, when RN-NPs are digested by SIF, the absolute value of the Zeta potential decreases significantly. This change indicates that the surface electrical properties of RN-NPs change in the intestinal environment, affecting their stability and biodistribution in the intestine.

[0066] The morphological changes of RN-NPs in SGF and SIF were observed by optical microscopy. Figure 6 Middle A, Figure 6 Middle B, Figure 6 Middle C, Figure 6 Medium D and Figure 6 As shown in Figure E, during the SGF digestion process, RN-NPs maintained a regular spherical structure without significant morphological changes, which further proves its stability in gastric juice. In the early stage of SIF digestion, the morphology of RN-NPs remained basically unchanged, except that the liposome membrane showed slight swelling and thickening; as the digestion process progressed, especially in the late digestion stage, some RN-NPs began to aggregate and began to show irregular flake or long strip morphology. This morphological change may be due to the gradual destruction of the liposome membrane by bile salts and trypsin in SIF. In summary, through the study of the average particle size, PDI, Zeta potential and microstructure changes of RN-NPs in the simulated gastrointestinal digestion process, it was found that it has good stability in gastric juice and can resist the influence of gastric acid and pepsin, so as to reach the intestine smoothly. In the intestinal environment, RN-NPs will gradually be emulsified by bile salts and hydrolyzed by trypsin, resulting in a decrease in particle size and morphological changes. The dispersibility of RN-NPs is still good throughout the digestion process, showing its potential application value as a drug carrier in the gastrointestinal tract.

[0067] The results of the digestion experiment show that the RN-NPs prepared in this study have good stability and can maintain their original physical and chemical properties for a long time. RN-NPs have strong gastric juice tolerance and can reach the intestine smoothly, and the digestion of RN-NPs in the intestine is slow and stable. This characteristic helps to achieve targeted drug release of RN-NPs in the intestinal tract, further improving the therapeutic efficiency of the drug.

[0068] 5. The RN-NPs nanoliposomes provided in Example 1 were subjected to an in vitro pore-forming toxin-stimulated release mechanism study. The experimental scheme was as follows: the concentrations of Escherichia coli and Staphylococcus aureus were adjusted to 10 using phosphate buffered saline (PBS). 6CFU / mL, and RN-NPs were added to the bacterial test tube. It was cultured at 37°C, 150rpm, and the number of residual bacteria in different groups was measured at different time points. At the same time, the samples incubated for 24 hours were analyzed for Res and Nisin content using high performance liquid chromatography. The results are as follows Figure 7-8 .

[0069] The results are as follows Figure 7 Middle A, Figure 7 Middle B, Figure 7 Middle C, Figure 7 Middle D, Figure 7 Middle E, Figure 7 Medium F, Figure 7 Medium G and Figure 7 As shown in Figure H, during the entire experiment, when RN-NPs were incubated with E. coli, the number of E. coli remained at a stable stage, and its number did not show a significant decrease, which was consistent with the results of the blank control group. This is because E. coli cannot secrete PFT to form holes in the liposome membrane and cannot intelligently release RN from the liposome. In the Staphylococcus aureus group containing RN-NPs, the number of Staphylococcus aureus continued to decrease with the extension of the reaction time. After 72 hours of reaction between Staphylococcus aureus and RN-NPs liposomes, there were no colonies on the plate. This suggests that once α-hemolysin is inserted into the bilayer of the liposome membrane, a pore structure will be formed on the membrane surface, and RN will be released from the liposome and react to Staphylococcus aureus. The experimental results accurately prove that α-hemolysin can be combined with RN-NPs to establish an intelligent triggering mechanism, thereby achieving the purpose of α-hemolysin stimulating RN-NPs to respond and release RN.

[0070] As mentioned above, after α-hemolysin reacts with liposomes, pores are formed on the liposome membrane, and RN is released from the liposomes. Using this feature, the reaction liquids after Escherichia coli and Staphylococcus aureus reacted with RN-NPs were collected for high-performance liquid chromatography. If Res and Nisin are present, the stimulated release mechanism can be proved. Figure 8 As shown in the figure, no chromatographic peaks of Res and Nisin appeared in the chromatogram of blank liposomes, indicating that there was no free Res and Nisin in the suspension. The same result was found in the reaction solution after E. coli and RN-NPs reacted, and no Res and Nisin were detected. This is because E. coli itself does not produce PFT, so it cannot release Res and Nisin from liposomes through intelligent response. In contrast to these results, Res and Nisin were detected in the reaction solution after Staphylococcus aureus and RN-NPs reacted, indicating that the a-hemolysin of Staphylococcus aureus can stimulate RN-NPs to release Res and Nisin.

[0071] 6. The RN-NPs nanoliposomes provided in Example 1 were tested for in vitro anti-inflammatory activity, and the test scheme was as follows: The cytotoxicity of the nanoparticles was determined by cell thiazole blue (MTT) colorimetry. Mouse macrophages J774A.1 were incubated overnight in a 96-well plate at 37°C and 5% CO2 for standby use. The cells were washed with Hanks buffer (HNSS) preheated to 37°C. While setting up a blank group, 100 μL of nanoparticles with different concentration gradients (0.01-5 mg / mL) were added to each well and incubated at 37°C for 4h. Subsequently, 0.5 mg / mL MTT solution was added to each well and incubated at 37°C for 3 hours. After removing the excess MTT solution, 200 μL DMSO was added to form blue-purple crystals. The absorbance at 560nm was measured using an enzyme reader and the cell survival rate was calculated.

[0072] Fresh mouse blood was used to conduct the hemolytic test of RN-NPs, and the international hemolytic standard of 5% was used as the evaluation standard for the experiment. PBS was used as the negative control and water was used as the positive control, and the absorbance of different media was measured at 570nm. The calculation formula is as follows:

[0073]

[0074] The nanoparticles were tested for their in vitro anti-inflammatory activity using J774A.1 cells. 105 cells were seeded into a 48-well plate per well and incubated overnight. The cells were rinsed with HBSS solution preheated at 37°C for later use, and 300 μL of LPS at a concentration of 100 ng / mL was added to each well and incubated for 20 hours. After the incubation, 30 μL of nanoparticles of different concentrations were added and incubated for 4 hours. The RN-NPs were crushed to release the RN encapsulated therein. The supernatant from each well was collected and centrifuged to remove cell debris. The TNF-α content was determined using an ELISA (Invitrogen, USA) quantitative kit, and the total protein content was determined using a BCA protein detection kit. Results Figure 9-10 .

[0075] Depend on Fig. 9 It can be seen that in the model group, LPS stimulation caused J774A.1 cells to synthesize and release a large number of inflammation-related proteins, which shows that the modeling method is successful and meets the requirements of subsequent experiments. Fig. 9As shown in A, after co-culturing blank nanoliposomes with LPS-stimulated J774A.1 cells at different concentrations, it was found that the protein content did not decrease significantly, indicating that blank nanoliposomes do not have anti-inflammatory effects. After RN-NPs treatment, the protein content produced by J774A.1 cells decreased significantly with the increase in the concentration of RN-NPs. When the concentration of RN-NPs increased to 4 mg / mL, the protein content decreased by about 82% compared with the blank nanoliposome group. This result shows that RN-NPs have an effective anti-inflammatory effect in vitro.

[0076] Depend on Fig. 9 As shown in B, the TNF-α level of J774A.1 cells in the model group increased significantly after LPS stimulation, which corresponds to the fact that the aforementioned LPS stimulation caused J774A.1 cells to synthesize and release a large number of inflammation-related proteins. After incubation with blank nanoliposomes and RN-NPs, it was found that blank liposomes had almost no effect on the changes in TNF-α levels, which further verified that liposomes themselves did not have anti-inflammatory effects. However, the level of TNF-α in the experimental group decreased significantly with the continuous increase in the concentration of RN-NPs, which echoed the previous research conclusions and further demonstrated that RN-NPs can effectively block the amplification of inflammation mediated by proinflammatory cytokines. In summary, RN-NPs have significant in vitro anti-inflammatory effects, which can effectively inhibit the synthesis of J774A.1 cell-related proteins after LPS stimulation and significantly reduce the secretion of proinflammatory factor TNF-α. This finding provides strong experimental support for RN-NPs as a potential anti-inflammatory treatment and lays the foundation for its further application research.

[0077] Depend on Fig.10As shown in A, the cell survival rate after RN-NPs treatment was greater than 70% in all tested concentration ranges. According to the 70% cytotoxicity critical value recommended by ISO10993-5:2009 (Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test), this experiment proved that RN-NPs had less cytotoxic effect on J774A.1 cells and were relatively biocompatible at various concentrations. This result may be related to the characteristics of Res and Nisin themselves. As a natural dietary polyphenol, Res has great health benefits for the intestine, and these beneficial effects are mainly related to its physiological activity in the gastrointestinal tract (GI). When Res is ingested, it is rapidly metabolized by the host and intestinal microbial metabolic enzymes to produce glucuronidated and sulfated metabolites. Among them, resveratrol-3-O-sulfate, in particular, has excellent regulatory functions on intestinal microbial growth and intestinal barrier function. In addition, Nisin can be degraded by digestive enzymes in the body without producing toxic substances, and will not cause significant negative effects on intestinal flora. Nisin has been approved for use as a food additive in many countries due to its safety and strong antibacterial activity. Therefore, RN-NPs have strong biosafety and can be used in subsequent experiments.

[0078] Depend on Fig.10 As shown in Figure B, water can cause hemolysis of blood, with a hemolysis rate of up to 100%, while PBS basically does not cause hemolysis. Fig.10 As shown in Figure B, when the concentration of RN-NPs is in the range of 0-6 mg / mL, the hemolysis rate caused by RN-NPs is lower than the internationally recognized standard of 5%. This shows that there is no obvious hemolysis in the blood of mice treated with RN-NPs, and it has good biocompatibility. It can be seen that RN-NPs have good biocompatibility and have great application potential in subsequent clinical applications.

[0079] 7. The RN-NPs nanoliposomes provided in Example 1 were subjected to an in vivo efficacy study. The test protocol was as follows: an antibiotic mixture consisting of kanamycin (0.6 mg / mL), gentamicin (0.0525 mg / mL), colistin sulfate (1275 U / mg), metronidazole (0.3225 mg / mL), vancomycin (0.0675 mg / mL) and cefaclor (0.45 mg / mL) was added to the drinking water of mice for 4 days of pretreatment. The mice were inoculated with 10 7 CFU Staphylococcus aureus. On day 11, mice were given free RN or RN-NPs or positive drug oxacillin sodium by gavage. The administration lasted for 6 days, and the mice were killed on day 18.

[0080] Mouse body weight and clinical activity score: Clinical activity score was performed on each mouse in each group. The score (0-4) was calculated using weight loss, stool consistency and rectal bleeding.

[0081] Myeloperoxidase (MPO) activity: After the mice were dissected and their intestines were removed, they were immediately quickly frozen with liquid nitrogen and kept at -80°C. Accurately weigh the animal tissue and add 9 times the volume of homogenization medium (0.9% saline) at a ratio of weight (mg): volume (μL) = 1:9. Mechanically homogenize under ice-water bath conditions to prepare a 10% homogenate, centrifuge at 2500-3000 rpm for 10 minutes, and take the supernatant for measurement. Each group was measured using the MPO kit (A044-1, Nanjing Jiancheng). Incubate in a 60°C water bath for 10 minutes, then immediately read the absorbance at a wavelength of 460nm, 1cm optical path, and zero with distilled water.

[0082] Tissue cytokine quantification: After collecting the intestinal tissue supernatant of the experimental mice, the content of IL-6 and IFN-γ was determined using Mouse IL-6 ELISA Kit and Mouse IFN-γ ELISA Kit (Invitrogen, USA).

[0083] Histological evaluation: The samples were fixed with 4% paraformaldehyde solution overnight (12h), and the histological evaluation was performed after good fixation. The tissue sections were fixed and embedded in paraffin. Two groups of three 10μm sections were cut with an interval of 150μm. The sections were stained with hematoxylin-eosin (H&E). The histological damage score was performed in a blinded manner, and the histological damage score was expressed as a percentage of the total tissue damage. The results are shown in Figure 11-15 .

[0084] Depend on Fig.12 As shown in A, the weight of healthy mice in the blank group gradually increased throughout the experiment, while the weight of all mice fed with the antibiotic mixture decreased. After the first day of inoculation with Staphylococcus aureus, the weight of mice in each experimental group increased, which may be due to the gradual decrease in the effect of antibiotics, and the number of Staphylococcus aureus colonization was not enough to cause a significant disease response. After drug treatment, the weight of mice in all experimental groups showed a recovery trend. Among them, the weight recovery effect of mice in the positive drug group (oxacillin sodium) was relatively poor. After stopping antibiotic feeding and inoculating Staphylococcus aureus, the weight of mice increased but the increase rate was relatively slow, indicating that the positive drug oxacillin sodium has a certain lag in the treatment of Staphylococcus aureus enteritis and the treatment effect is general. In contrast, the weight of mice treated with free RN and RN-NPs recovered rapidly, especially the weight of mice in the RN-NPs group increased the fastest, proving that RN-NPs can play a faster role and have the best recovery effect.

[0085] S. aureus infection did not cause strong clinical symptoms of the disease, nor was there any abnormal behavioral changes or explosive diarrhea in the mice. Only soft and sticky feces were observed in the infected mice. Fig.12 As shown in Figure 3B, the DAI score of the enteritis model group maintained an upward trend, indicating that the severity of enteritis continued to increase. As the administration time prolonged, the DAI scores of mice in other groups decreased. Although positive drugs show certain efficacy in treatment, their effects are delayed. In the free RN group and RN-NPs group, the DAI score began to decline significantly from the second day of administration, especially in the RN-NPs group, where the score decline was more significant. This shows that the therapeutic effect of RN-NPs is stronger than that of free RN and positive drug groups. In summary, compared with the positive drug treatment group and free RN, RN-NPs can not only restore the weight of mice faster, but also significantly reduce the DAI score, proving that it has a strong anti-inflammatory effect and better clinical application prospects.

[0086] like Fig.12 C and Fig.12 As shown in D, the levels of IFN-γ and IL-6 in mice in the antibiotic group were higher than those in normal mice, but significantly lower than those in the model group. This may be due to the fact that the colonization of Staphylococcus aureus exacerbated the severity of inflammation in mice. After drug treatment, the levels of IFN-γ and IL-6 in mice decreased. Specifically, the inhibitory effects of free RN and RN-NPs were significantly better than those of the positive control group. Compared with the free RN group, RN-NPs treatment can more significantly reduce the expression levels of inflammatory cytokines IFN-γ and IL-6. This further proves the anti-inflammatory advantages of RN-NPs.

[0087] Depend on Fig.12 As shown in E, the MPO activity of healthy mice in the blank group was within the normal range, but the MPO activity in the model group increased significantly, indicating the production of inflammation in mice in the model group. Compared with the model group, the positive control group, free RN group and RN-NPs group significantly inhibited the increase in MPO activity. After the treatment, the MPO content in the RN-NPs group decreased by about 52% and 28% compared with the positive control group and the free RN group, respectively. It can be seen that RN-NPs showed significant anti-inflammatory effects in reducing the levels of inflammatory cytokines IFN-γ and IL-6 and inhibiting MPO activity, further demonstrating its potential as an anti-inflammatory therapeutic agent.

[0088] H&E staining was used to observe the effect of RN-NPs on the morphology of mouse colon tissue and the severity of the disease was scored histologically. Fig.13As shown in A, B, C, D, E, F, G, H, I, J, K, and L, the normal group showed that the healthy mice had abundant intestinal folds, a large number of closely arranged intestinal glands in the mucosal epithelial lamina propria, abundant goblet cells, loose connective tissue in the submucosal layer, and a clear muscular layer structure. In the model group, the intestinal mucosal epithelial cells were occasionally edematous, the intestinal glands were slightly dilated, and more granulocytes and lymphocytes were infiltrated in the lamina propria. Similarly, necrosis and shedding of mucosal epithelial cells were observed in the antibiotic group, and a small amount of granulocyte infiltration was observed in the lamina propria. However, in the other experimental groups treated with drugs, the intestinal inflammation of mice was alleviated. Among them, compared with the model group, the symptoms of the positive control group were slightly alleviated, but a small amount of lymphocytes and granulocytes were still observed infiltrating, the mucosal epithelium was separated from the lamina propria, and a small range of loosely arranged muscle cells were observed in the muscular layer. It is worth mentioning that in the free RN group, a small amount of necrotic and detached mucosal epithelial cells were found in the intestinal cavity, and occasional focal aggregation and infiltration of lymphocytes were found, indicating that the therapeutic effect of free RN on intestinal inflammation is limited. In the RN-NPs group, the number of colonic folds was abundant, the crypt depth increased, and the distance between the bottom of the crypt and the mucosal muscle decreased. There were abundant goblet cells, the lamina propria contained a large number of intestinal glands, and the intracellular mucus increased. A small area of ​​loosely arranged muscle cells was still visible in the muscular layer, and no obvious histological damage was observed. At the same time, Fig.14 It can be seen that the histological score of the RN-NPs group was reduced most significantly. It can be seen that RN-NPs has a significant therapeutic effect on Staphylococcus aureus enteritis.

[0089] like Fig.15As shown in A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W and X, the analysis of cardiac tissue sections showed that the myocardial fibers of the blank group (healthy mice) were uniformly colored, with obvious cell boundaries, consistent fiber arrangement, clear myocardial cell striated structure, and no abnormalities in the interstitium. However, the myocardial cells in the model group showed obvious pathological characteristics, with cell swelling, enlarged intercellular gaps, loose cytoplasm, and faded staining at the blue arrows. This phenomenon was alleviated after drug treatment. In particular, the therapeutic effect of RN-NPs was the most prominent, and it can be seen that the myocardial cells were arranged more orderly, and the overall density of the myocardial tissue was restored. At the same time, through the observation of liver tissue, it was found that compared with the normal liver of the blank group, the gaps between the hepatocytes in the model group became larger, and the liver showed edema. However, after treatment with positive drugs and free RN, the recovery of the liver sinusoids was not significant. However, in the RN-NPs treatment group, it was found that the intercellular gaps of the hepatocytes were significantly improved, and the edema phenomenon was also alleviated. In the histological analysis of the spleen, the spleen of healthy mice showed clear boundaries between white pulp and red pulp, distinct marginal areas, and intact lymph nodule structure. In the model group, the white pulp structure of the spleen tissue was damaged and the marginal area was blurred. After drug treatment, the structural damage of the spleen was improved, especially in the RN-NPs treatment group. In the observation of kidney tissue, no obvious damage or lesions were found in all groups, indicating that the kidneys were not significantly affected under the experimental conditions. In summary, RN-NPs did not show adverse effects on other organs in the treatment of enteritis in mice, and it was effective in protecting the myocardium, liver, and spleen.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing nanoliposomes, characterized in that: The steps include: S1. Dissolve soybean lecithin, cholesterol and Tween 80 in anhydrous ethanol, stir until completely dissolved, and transfer the mixed solution to a rotary evaporator to obtain a lipid film; S2, adding resveratrol solution, nisin solution and 0.03 M phosphate buffer to the lipid film obtained in step S1, and transferring the film to a cell ultrafine pulverizer for treatment to obtain a nanoliposome solution after the film is completely dissolved; S3. Add 3% by mass of phosphatidylethanolamine-polyethylene glycol 2000 aqueous solution to the nanoliposome solution obtained in step S2, vortex and mix evenly, let stand, and filter with a filter membrane to obtain polyethylene glycol-modified nanoliposome RN-NPs.

2. The preparation method according to claim 1, characterized in that: In step S1, the weight ratio of soybean lecithin, cholesterol and Tween 80 is 6:1:1.

8.

3. The preparation method according to claim 1, characterized in that: In step S1, the mixed solution is transferred to a rotary evaporator and heated in a water bath at 50-60° C. for 5-15 min to obtain a lipid film.

4. The preparation method according to claim 1, characterized in that: In step S2, the pH of the phosphate buffer is 7.2-7.

4.

5. The preparation method according to claim 1, characterized in that: In step S2, the cell ultrafine pulverizer processing parameters are: working for 10 seconds, interval of 5 seconds, power of 300W and working for 30 minutes.

6. The preparation method according to claim 1, characterized in that: In step S3, after vortexing to mix evenly, the mixture was allowed to stand at 4° C. for 1 h.

7. The preparation method according to claim 1, characterized in that: In step S2, the filter membrane is a 0.22 mm microporous filter membrane.

8. The nanoliposome prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the nanoliposomes prepared by the preparation method according to any one of claims 1 to 7 or the nanoliposomes according to claim 8 in preparing a product for targeted treatment of Staphylococcus aureus enteritis.

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